The cornerstones for controlling infections caused by non-tuberculosis mycobacteria (NTM) are the macrolides, clarithromycin and azithromycin. The genetic basis of clinically acquired resistance to these agents is conferred by mutation in the 23S ribosomal RNA (rRNA) gene. However, we understand little of the mechanisms of intrinsic macrolide resistance of mycobacteria, particularly M. tuberculosis. Recent studies suggest that mycobacteria have erm methylase genes and macrolide efflux pumps, which may affect susceptibility to macrolides. Therefore, we hypothesize that there are several mechanisms that affect the antimycobacterial activity of macrolides. These mechanisms include drug efflux, expression of erm genes, and, the acquisition of a 23S rRNA gene mutation. Consequently, the long-term objective of this work is to characterize the mutation-independent mechanisms that affect mycobacterial susceptibility to macrolides. To address this objective, this project is divided in to 3 specific aims: (1) to identify the genes conferring inducible macrolide resistance; (2) to investigate the macrolide efflux systems of mycobacteria; and, (3) to characterize the prevalence of resistance genes within the Mycobacteriaceae. Understanding the processes that affect the antimycobacterial activity of macrolides will directly impact the development of new drugs and improve treatment regimens. Of particular interest is improving the anti-tuberculosis activity of macrolides.